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Highly active, citrate inhibition resistant form of Aspergillus niger 6-phosphofructo-1-kinase encoded by a modified pfkA gene

Paper ID Volume ID Publish Year Pages File Format Full-Text
24469 43517 2009 7 PDF Available
Title
Highly active, citrate inhibition resistant form of Aspergillus niger 6-phosphofructo-1-kinase encoded by a modified pfkA gene
Abstract

In Aspergillus niger cells spontaneous posttranslational modification of 6-phosphofructo-1-kinase (PFK1) occurs. In a two step process the native enzyme (85 kDa) is first cleaved to an inactive fragment (49 kDa) that regains its activity after phosphorylation of the protein. The shorter PFK1 fragment exhibits changed kinetics, such as resistance to citrate inhibition. In order to avoid spontaneous complex posttranslational modification, modified gene was prepared encoding an active shorter PFK1 fragment. Since no appropriate microbial strains with disrupted native pfkA genes were available, Aspergillus niger strain with reduced likelihood for spontaneous posttranslational modification of PFK1 has been chosen for in vivo tests. First, the appropriate length of a truncated gene was defined after a number of enzymes encoded by genes of different lengths had been tested. After adding sodium azide to the medium, phosphorylation was induced in the transformed hyphae to activate the shorter fragments which were subsequently screened for changed PFK1 kinetics. In the second step the responsible threonine residue was replaced with glutamic acid to elude the need for phosphorylation. An active shorter PFK1 fragment, resistant to citrate inhibition and activated to a higher level by fructose-2,6-bisphosphate with respect to the native enzyme was encoded directly from the modified gene.

Keywords
Aspergillus niger; 6-Phosphofructo-1-kinase; Modified gene; Citrate resistance; Citric acid
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Highly active, citrate inhibition resistant form of Aspergillus niger 6-phosphofructo-1-kinase encoded by a modified pfkA gene
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 144, Issue 1, 12 October 2009, Pages 51–57
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us